The proper intracellular distribution of lipids is vital for many cellular functions. Relatively little is known, however, about the means by which cells determine the lipid composition of organelles and regulate intracellular lipid transport. Numerous diseases, including heart disease and Type 2 diabetes, are associated with defects in these processes. The lipid composition of organelles is determined by both modulating the lipid composition of intracellular transport vesicles and nonvesicular lipid transport between organelles. Neither process is well understood. We seek to identify proteins required for nonvesicular lipid trafficking in cells and understand their role in organelle biogenesis and lipid metabolism. Identification of these proteins is critical for understanding organelle biogenesis and how defects in lipid transport contribute to many diseases. To this end, we use a combination of biochemical and genetic approaches in the model organism S. cerevisiae. We are also studying a novel nuclear import pathway used by a transcription factor that regulates lipid metabolism. 1. Oxysterol binding proteins and PIPs are required for non-vesicular sterol transport from the plasma membrane to the ER. The proper intracellular distribution of sterols such as cholesterol is critical for numerous cellular functions, including signal transduction and protein trafficking. Sterols are moved among cellular compartments by both vesicular and poorly understood nonvesicular pathways. We have previously shown that a non-vesicular pathway moves sterols from the plasma membrane (PM) to the endoplasmic reticulum in the yeast S. cerevisiae. We found that this transport requires oxysterol-binding proteins (OSBPs), a large family of lipid-binding proteins that is conserved from yeast to humans. In addition, we have been able to show that some of these yeast OSBPs transport sterols and other lipids in vitro. Thus, lipid transport is one of the functions of this important class of proteins. We also found that sterol transport by OSBPs is regulated by particular phosphoinositides (PIPs). Because different PIP-species are enriched in various cellular compartments, PIP-stimulation of ORPs likely serves to regulate the movement of sterols (and possibility other lipids) to particular organelles by ORPs, perhaps in response to cell signaling events. This work is described in a paper that is under review. 2. Identification of a RAN-independent, calmodulin-dependent nuclear localization signal (NLS) in the high mobility group (HMG) transcription factor Nhp6ap. We have identified the NLS required for the RAN-independent import of Nhp6ap and demonstrated that it can bind calmodulin, a protein that may play a role in a novel nuclear import pathway. Mutants with calmodulin defects fail to localize a protein containing the Nhp6ap NLS to the nucleus. These mutants do not affect the localization of a reporter with a RAN-dependent NLS. Thus, Nhp6ap is imported into the nucleus by a novel, calmodulin-dependent, RAN-independent nuclear pathway. These results are described in a manuscript that is being prepared. We have isolated a mutant with a defect in this novel import pathway. Characterization of this mutant should reveal other components of this pathway. 3. Reconstitution of nonvesicular lipid transport from the ER to peroxisomes and mitochondria. Peroxisomes, and mitochondria play critical roles in lipid homeostasis and other vital cellular functions. Since there is no known vesicular transport between these organelles and other compartments in the cell, they must acquire lipids by nonvesicular mechanisms. We have reconstituted nonvesicular lipid transport to these compartments in vitro. In all cases, we find that there is rapid, nonvesicular lipid transport between ER-derived vesicles and these organelles. This transport does not require cytosol or ATP, but does require proteins on the ER and the target organelle. We are in the process of identifying proteins needed for these novel lipid transport pathways.